Local probe microscopy on lipid membranes: near field optical imaging and shear force studies

Many fundamental processes that govern the function of biological cells occur on or immediately under the cell membrane. Fluorescence marking of specific sites has had a large impact in the recognition of these processes. In addition, observation in an appropriate ionic buffer is required in many cases to preserve a reliable dynamics of the cell. Artificial membranes, used as a model for biological ones, can provide fundamental parameters connected to the composition and the molecular organization in natural membranes. Near field optical microscopy offers a valid possibility to obtain spectroscopic information at subwavelength scale, performing observation also in native liquid environment. This thesis is focused on the study and the improvements of different aspects of this technique to the observation of fluorescently labelled thin lipid films in different environments. More specifically, three fundamental issues have been addressed in this work: the implementation of a homemade SNOM in liquid, the near field fluorescence observation of Langmuir-Blodgett monolayers, and the study of probe-sample interaction forces on flat lipid films. Near field imaging in water is a critical instrumental aspect due to high drag forces generated in the liquid medium by the vibrating tip. These forces, that can induce a significant indentation of a compliant sample, have been minimized by a specific set up. The near field probe minimally interacts with the surrounding medium, being protected by a capillary tube. This dispositive has allowed measurements on thin lipid films immersed in water. However, measurements on fixed cell in water have induced indentation of the sample. The boundary transition width of non fluorescent liquid condensed domain surrounded by a liquid expanded fluorescent matrix, in a thiolipid Langmuir-Blodgett monolayer, has been studied in the near field. The optical image gives interesting insights on the molecular organization within the liquid condensed domain, showing in the central part a different molecular organization with respect to the rest of the domain. Force mapping of the sample in air predicts a contrast between the two phases that does not correspond to the topological relief of the film. Measurements in water show an inverted contrast with respect to the one recorded in air. To better identify the different forces that contribute to the shear force contrast when scanning a lipid film, the dynamics of Al-coated and probes bearing chemically functional groups has been examined in the interaction with two different systems. Interfacial forces on self assembly monolayer, micropatterned with molecules exhibiting opposite water affinity, have been investigated with Al-coated and hydrophobic functionalized near field probe. These experiments indicate that the tip dynamics in the sample vicinity is critically dependent on the wettability properties of both interfaces. The contrast observed when scanning a thiolipid monolayer is, also in this case, tightly linked with the surface properties of the probe. In particular, hydrophilic probes are totally insensitive to the different packing of alkyl chains in the two phases. The dynamics of fluorinated surface probes shows the very interesting feature of not being influenced by the drag forces of the surrounding liquid medium. This result opens up the possibility to employ such a tip, whose mechanical properties are equivalent in air and in water, to scan very compliant samples in liquid environment.